The session got off to a fast start thanks to the Michelin rubber left on the track by the Carrera Cup cars.
Brodie Kostecki was initially fastest with a 1m06.591s before being jumped by Bryce Fullwood's 1m06.509s.
Eight minutes into the half-hour session Cam Waters lowered the benchmark to a 1m06.471s, only to be immediately beaten by Anton De Pasquale, who stopped the clock at 1m06.285s.
That time proved to be unbeatable, the DJR driver leading a trio of Mustangs at the flag.
"We've had a couple of good sessions, which is really good," said De Pasquale.
"Darwin has always been good to us, so this is good place if we're going to turn it around.
"We had a really good test day a couple of weeks ago, a good last race in Tassie, not a good result but some stuff learned that we took forward. We're a lot better than we were a few weeks ago."
It was a solid session for Tickford, James Courtney ending up second, just three-hundredths behind De Pasquale, while Waters ended up third.
Brodie Kostecki was the best-placed Camaro in fourth ahead of Thomas Randle, Andre Heimgartner, David Reynolds and Fullwood.
Will Davison was ninth and Macauley Jones 10th in what is the final on-track session ahead of qualifying tomorrow.
Van Gisbergen, meanwhile, had another tough session as he looked to recover from a significant loss of running in practice 1.
The Kiwi spent the first half of the session complaining about both a tracking issue and poor throttle response. At one point he told the team, "throttle response is completely fucked. I can't drive the car" over the radio.
Unable to make any engine changes under the new rules, the team attempted to fix the tracking issue with a little over 10 minutes to go. But after an installation lap van Gisbergen returned to the pits for the remainder of the session.
That left him second-last on the time sheets.
Triple Eight teammate Broc Feeney, who topped the morning session, wasn't particularly speedy in practice 2 either, setting just the 12th fastest time.
Van Gisbergen had good company at the bottom of the times, with Chaz Mostert ending up dead last.
The Walkinshaw Andretti United driver didn't get a clean green tyre run all session, nipping a brake on his crucial final flyer.
The Darwin Triple Crown continues with qualifying for race 1 at 11:30pm local time tomorrow.
